R-hydroxynitrillyases havign improved substrate tolerance and the use thereof

ABSTRACT

The invention related to R-hydroxynitrillyases from the family of Rosaceae that are characterized by an improved substrate tolerance and increased stability. In the active center of the R-hydroxynitrillyases either a) an alanine group is substituted by glycine, valine, leucine, isoleucine, or phenylalanine or b) a phenylalanine group is substituted by alanine, glycine valine, leucine or isoleucine, or c) a leucine group is substituted by alanine, glycine, valine, isoleucine or phenylalanine, or d) an isoleucine group is substituted by alanine, glycine, valine, leucine or phenylalanine. The invention also relates to the use of these lyases in the production of enantiomer-pure R- or S-cyanohydrines.

Biocatalytic processes have become very important for the chemicalindustry. The carrying-out of chemical reactions with the assistance ofbiological catalysts is in this connection of interest especially inareas of application in which it is possible to exploit the property ofenzymes, which is often present, of preferentially converting or formingone of the two enantiomers in chemical reactions with chiral orpro-chiral components.

Essential preconditions for exploiting these favorable properties ofenzymes are their availability in the quantities required industriallyand a sufficiently high reactivity, as well as stability under theactual conditions of an industrial process.

A particularly interesting class of chiral chemical compounds arecyanohydrins. Cyanohydrins are important for example in the synthesis ofα-hydroxy acids, α-hydroxy ketones, β-amino alcohols, which are used forobtaining biologically active substances, e.g. active pharmaceuticalingredients, vitamins or pyrethroid compounds.

These cyanohydrins are prepared by addition of hydrocyanic acid onto thecarbonyl group of a ketone or aldehyde.

It has been possible to achieve the industrial preparation of chiralcompounds such as, for example, (S)-cyanohydrins by making the enzyme(S)-hydroxynitrile lyase from Hevea brasiliensis available, as describedfor example in WO 97/03204, EP 0 951561 and EP 0 927 766.

However, there is a multiplicity of interesting chemical compounds forwhich the R enantiomers are important for industrial applications. Todate, only processes for preparing a number of products which can beemployed only on the laboratory scale have been described (e.g.: EP 0276 375, EP 0 326 063, EP 0 547 655). The enzyme preparations employedin these cases were mainly those obtained from plants of the Rosaceaefamily, for example from almond kernels (Prunus amygdalus).

Further R-HNLs which have been employed to date are, for example, thosefrom linseed seedlings (Linum usitatissimum; LUHNL) which were cloned asfirst gene of an R-HNL and were expressed in E. Coli and Pichiapastoris, or R-HNL from Phlebodium aureurum.

Industrial applications on a larger scale have not been achieved todate. The essential reason for this is that enzyme preparations fromplants of the Rosaceae family having hydroxynitrile lyase (HNL) activityor those from linseed seedlings have not to date been available insufficient quantities and at reasonable costs and, moreover, showed astability which was too low at low pH values.

Advantageous reaction parameters described in the literature forobtaining products with high optical purity are low temperatures (e.g.Persson et al.; Enzyme and Microbial Technology 30(7), 916-923; 2002), apH below 4 (e.g. Kragl et al.; Annals of the New York Academy ofScience; 613 (enzyme Eng. 10), 167-75, 1990), and the use of 2-phasesystems (for example EP 0 547 655) or of emulsions (e.g. EP 1 238 094).Unfortunately, most R-HNLs have half-lives of less than one hour at a pHbelow 4.

EP 1223220 A1 describes recombinant enzymes which are prepared bycloning a gene from Prununs amygdalus, which codes for an R-HNLisoenzyme, for example for isoenzyme 5 (PaHNL5), and by heterologousexpression for example in Pichia pastoris, which are distinguished, asis evident from the examples, by a considerably increased stability atlow pH values compared with the other known R-HNLs.

A disadvantage which has been found is that the substrate acceptance isunsatisfactory, because conversion of some substrates in the presenceof, for example, recombinant PaHNL5 takes place at a distinctly lowerreaction rate than in the presence of commercially available vegetable,native (R)-HNL preparations from almond kernels.

It was therefore an object of the invention to provide R-hydroxynitrilelyases from the Rosaceae family which firstly can be provided on asufficient scale and cost-effectively for technical conversions on theindustrial scale, and which display an improved substrate acceptance andan increased stability.

The invention accordingly relates to R-hydroxynitrile lyases from theRosaceae family having improved substrate acceptance and increasedstability, which are characterized in that in the active center of theR-hydroxynitrile lyases there is replacement either of

-   -   a) an alanine residue by glycine, valine, leucine, isoleucine or        phenylalanine or    -   b) a phenylalanine residue by alanine, glycine, valine, leucine        or isoleucine or    -   c) a leucine residue by alanine, glycine, valine, isoleucine or        phenylalanine or    -   d) an isoleucine residue by alanine, glycine, valine, leucine or        phenylalanine.

The R-HNLs of the invention are mutants of R-hydroxynitrile lyases fromthe Rosaceae family.

It is possible to employ as initial basis for preparing the mutants ofthe invention native R-HNLs from the Rosaceae family, such as, forexample, R-HNLs from Prunus amygdalys (PaHNL), Prunus serotina (PsHNL),Prunus laurocerasus, Prunus lyonii, Prunus armaniaca, Prunus persica,Prunus domestica (PdHNL), Malus communis, etc. or recombinant R-HNLs, asdisclosed for example in EP 1223220, and so-called tunnel mutants of theabovementioned R-HNLs, in which one or more bulky amino acid residueswithin the hydrophobic channel leading to the active center are replacedby less bulky amino acid residues.

The native R-HNLs which are preferably employed are R-HNLs from Prunusamygdalys (PaHNL), Prunus domestica (PdHNL) or from Prunus serotina(PsHNL).

Preferred recombinant R-HNLs are recombinant RHNLs from Prunus domestica(PdHNL), in particular PdHNL1, and the recombinant R-HNLs PaHNL1 toPaHNL5 which are described in EP 1223220, with particular preference forrecombinant PaHNL5.

Suitable tunnel mutants are preferably native or recombinant R-HNLs inwhich preferably one bulky amino acid residue in the hydrophobic channelleading to the active center has been replaced by a less bulky aminoacid residue such as, for example, alanine, glycine, valine orphenylalanine.

The R-HNLs to be modified may moreover be in the form of an alteredsequence which is obtained for example by exchange of the first aminoacid(s) in the sequence or by deletion of the first amino acid(s) or byattachment of further amino acids, such as, for example, GluAlaGluAla.

A further possibility before the mutation in the active center is toexchange the natural or vegetable signal sequence for another signalsequence such as, for example, for the signal sequence of the alphamating factor from Saccharomyces cerevisiae (alpha-MF), Saccharomycescerevisiae invertase (SUC2), Pichiakiller toxin signal sequence,α-amylase, Pichia pastoris acid phosphatase (PHO1), Phaseolus vulgarisagglutinin (PHA-E); glycoamylase signal sequence from Aspergillus niger(glaA), glucose oxidase (GOX) signal sequence from Aspergillus niger,Sec10 signal sequence from Pichia pastoris, signal sequence of the 28 kDsubunit of the killer toxin from Klyveromyces lactis, BSA signalsequence, etc., or a recombinant signal sequence thereof. The signalsequences may moreover comprise point mutations.

Suitable signal sequences and their mutants are described for example inHeijne G. et al., FEBS Letters 244 (2), 439-46 (1989), EP 19911213,Paifer et al., Biotecnologia Aplicada 10(1), 41-46, (1993), Raemaekerset al., European Journal of Biochemistry 265(1), 394-403 (1999) etc.

The vegetable signal sequence is preferably replaced by the signalsequence of the alpha mating factor from Saccharomyces cerevisiae.

The R-HNLs of the invention are prepared by site-specific mutagenesis,for example using the Quick Change (XL) Site Directed Mutagenesis Kit,Quick Change Multi Site Directed Mutagenesis Kit (from Stratagene), andkits from Invitrogen or Promega etc. in accordance with themanufacturer's instructions or by other conventional methods asdescribed for example in Current Protocols in Molecular Biology, Ausubelet al., 2003.

Site-directed mutagenesis kits are systems ready for use for preparingspecific mutants and are sold commercially for example by StratageneCloning Systems, La Jolla, CA (USA).

In the site-specific mutagenesis, there is according to the inventionreplacement in the active center of the R-HNL either of

-   -   a) an alanine residue by glycine, valine, leucine, isoleucine or        phenylalanine or    -   b) a phenylalanine residue by alanine, glycine, valine, leucine        or isoleucine or    -   c) a leucine residue by alanine, glycine, valine, isoleucine or        phenylalanine or    -   d) an isoleucine residue by alanine, glycine, valine, leucine or        phenylalanine.

In the mutants resulting therefrom the abovementioned residues, whichare located in the active center in the direct vicinity of thesubstrate-binding site, are exchanged.

It is preferred in this connection for there to be replacement either ofan alanine residue by glycine, valine or leucine or of a phenylalanineresidue by alanine, leucine or glycine or of a leucine residue byalanine, glycine or phenylalanine.

For conversion of large substrates such as, for example, of aromaticaldehydes or ketones having bulky radicals or with substituents in theortho or meta position, or of bulky aliphatic aldehydes or ketones,preferably one of the amino acids alanine, valine, leucine, isoleucineor phenylalanine is replaced by a smaller amino acid residue in eachcase. Thus, for example, phenylalanine is replaced by leucine oralanine; or alanine by glycine etc.

For conversion of smaller substrates such as, for example, smalleraliphatic aldehydes or ketones, by contrast, preferably one of the aminoacids alanine, valine, leucine, isoleucine or phenylalanine is replacedby a larger amino acid residue in each case, such as, for example,alanine by leucine or valine, or leucine by phenylalanine.

Particular preference is given to mutants of the recombinantR-hydroxynitrile lyases PaHNL1-PaHNL5 which are disclosed for example inEP 1223220 and which may optionally also have a mutation in thehydrophobic channel leading to the active center.

Especial preference is given to mutants of the recombinantR-hydroxynitrile lyase PaHNL5, which may optionally also have a mutationin the hydrophobic channel leading to the active center, in which thereis replacement in the active center of

-   -   a) the alanine residue in position 111 by glycine, valine or        leucine or    -   b) the phenylalanine residue at position 72 by alanine or        leucine or    -   c) the leucine residue at position 331 or 343 by alanine or        glycine.

The numberings are derived from the corresponding positions in themature unmodified recombinant R-hydroxynitrile lyase PaHNL5, but thepositions can be shifted according to the abovementioned modificationsof the sequence, such as, for example, truncation or extension of thesequence.

Secretory expression in suitable microorganisms then takes place, suchas, for example, in Pichia pastoris, Saccharomyces cerevisiae orEscherichia coli, Bacillus subtilis, Klyveromyces lactis, Aspergillusniger, Penicillium chrysogenum, Pichia methanolica, Pichia polymorpha,Phichia anomala, Schizosaccharomyces pombe etc.

The resulting R-HNL mutants of the invention are purified by standardmethods, for example in analogy to Dreveny et al.; Structure (Cambridge;MA, United States) 9(9), 803-815; 2001.

The R-HNL mutants of the invention are suitable for the preparation ofenantiopure R- or S-cyanohydrins in a conversion rate which is increasedcompared with the prior art, the R-HNL mutants of the invention alsobeing distinguished by a high pH stability at low pH values.

The invention accordingly relates further to the use of the R-HNLmutants of the invention for preparing enantiopure R- or S-cyanohydrins.

The R-HNL mutants of the invention are employed in particular withaliphatic and aromatic aldehydes and ketones as substrates.

Aldehydes mean in this connection aliphatic, aromatic or heteroaromaticaldehydes. Aliphatic aldehydes mean in this connection saturated orunsaturated, aliphatic, straight-chain, branched or cyclic aldehydes.Preferred aliphatic aldehydes are straight-chain or branched aldehydeshaving in particular 2 to 30 C atoms, preferably of 4 to 18 C atoms,which are saturated or mono- or polyunsaturated. The aldehyde may inthis connection have both C-C double bonds and C-C triple bonds. Thealiphatic, aromatic or heteroaromatic aldehydes may moreover beunsubstituted or substituted by groups which are inert under thereaction conditions, for example by optionally substituted aryl orheteroaryl groups, such as phenyl, phenoxy or indolyl groups, byhalogen, hydroxy, hydroxy-C₁-C₅-alkyl, C₁-C₅-alkoxy, C₁-C₅-alkylthio,ether, alcohol, carboxylic ester, nitro or azido groups.

Examples of preferred aliphatic aldehydes are butanal, 2-butenal,3-phenylpropanal, hydroxypivalaldehyde etc. Examples of aromatic orheteroaromatic aldehydes are benzaldehyde and variously substitutedbenzaldehydes such as, for example, 2-chlorobenzaldehyde,3-chlorobenzaldehyde, 4-chlorobenzaldehyde, 3,4-difluorobenzaldehyde,3-phenoxybenzaldehyde, 4-fluoro-3-phenoxybenzaldehyde,hydroxybenzaldehydes, methoxybenzaldehydes, also furfural,methylfurfural, anthracene-9-carbaldehyde, furan-3-carbaldehyde,indole-3-carbaldehyde, naphthalene-1-carbaldehyde, phthalaldehyde,pyrazole-3-carbaldehyde, pyrrole-2-carbaldehyde,thiophene-2-carbaldehyde, isophthalaldehyde or pyridinealdehydes,thienylaldehydes etc.

Ketones are aliphatic, aromatic or heteroaromatic ketones in which thecarbonylcarbon atom has different substituents. Aliphatic ketones meansaturated or unsaturated, straight-chain, branched or cyclic ketones.The ketones may be saturated or mono- or poly-unsaturated. They may beunsubstituted or substituted by groups which are inert under thereaction conditions, for example by optionally substituted aryl orheteroaryl groups such as phenyl or inolyl groups, by halogen, ether,alcohol, carboxylic ester, nitro or azido groups.

Examples of aromatic or heteroaromatic ketones are acetophenone,indolylacetone etc.

Aldehydes and ketones suitable according to the invention are known orcan be prepared in a conventional way.

The substrates are converted in the presence of the HNLs of theinvention with a cyanide group donor. Suitable as cyanide group donorare hydrocyanic acid, alkali metal cyanides or a cyanohydrin of thegeneral formula IR₁R₂C(OH)(CN).

In formula I, R₁ and R₂ are independently of one another hydrogen or anunsubstituted hydrocarbon group, or R₁ and R₂ together are an alkylenegroup having 4 or 5 C atoms, with R₁ and R₂ not both being hydrogen. Thehydrocarbon groups are aliphatic or aromatic, preferably aliphaticgroups. R₁ and R₂ are preferably alkyl groups having 1-6 C atoms, andthe cyanide group donor is very preferably acetone cyanohydrin.

The cyanide group donor can be prepared by known processes.Cyanohydrins, especially acetone cyanohydrin, can also be purchased.

The cyanide group donor employed is preferably hydrocyanic acid (HCN),KCN, NaCN, or acetone cyanohydrin, particularly preferably hydrocyanicacid. The hydrocyanic acid can moreover be liberated only shortly beforethe reaction from one of its salts such as, for example, NaCN or KCN andbe added undiluted or in dissolved form to the reaction mixture.

The conversion can be carried out in an organic, aqueous or 2-phasesystem or in emulsion, and without diluent.

An aqueous solution or buffer solution comprising the HNL of theinvention is used as aqueous system. Examples thereof are Na citratebuffer, phosphate buffer etc.

It is possible to use as organic diluent, water-immiscible or slightlywater-miscible aliphatic or aromatic hydrocarbons, which are optionallyhalogenated, alcohols, ethers or esters or mixtures thereof or thesubstrate itself. Methyl tert-butyl ether (MTBE), diisopropyl ether,dibutyl ether and ethyl acetate or mixtures thereof are preferablyemployed.

The HNLs of the invention can moreover be present either as such orimmobilized in the organic diluent, but the conversion can also takeplace in a two-phase system or in an emulsion with nonimmobilized HNL.

The conversion moreover takes place at temperatures of from −10° C. to+50° C., preferably at −5° C. to +45° C.

The pH of the reaction mixture can be from 1.8 to 7, preferably from 2to 4 and particularly preferably from 2.5 to 3.5.

EXAMPLE 1 Cloning of the pahnl4 gene from Prunus amygdalus

Gene-specific PCR primers based on the sequence homology of the mdl4gene of Prunus serotina were prepared:

The amplification took place in a 50 μl mixture with 1.2 U of “Hotstar”Taq DNA polymerase (Qiagen, Hilden, Germany), with 50 ng of genomicalmond DNA (isolated from Farmgold almond kernels batch number L4532,1999 harvest) as “template” and 10 pmol of each of the primers mandlp2f(oBT2204) and mandlp5r (oBT2206), 5 μl of DNTP (2 mM each) mix, all inlx PCR buffer in accordance with the manual of the “Hotstar Kit”(Qiagen, Hilden, Germany), starting with a 15-minute denaturation stepat 95 DEG C, followed by 10 cycles (1 min 94 DEG C, 1 min 45 DEG C; 1min 72 DEG C) for preamplification, 20 further cycles (1 min 94 DEG C, 1min 68 DEG C, 1 min 72 DEG C) for amplification of specific products anda final incubation at 72 DEG C for 5 min. A DNA fragment about 2.2 kb insize (determined by agarose gel electrophoresis analysis) was obtainedin this PCR. This PCR product was purified from an agarose gel using the“Qiaquick Kit” (Qiagen, Hilden, Germany) in accordance with the manualincluded, cloned via the EcoRI cleavage sites into the pBSSK(−) cloningvector, and sequenced using the “Dye Deoxy Terminator Cycle Sequencing”kit (Applied Bio-systems Inc., Forster City, CA, USA) by the primerwalking strategy. New PCR primers were derived from the 5′ and 3′regions of the DNA sequence and employed for a new PCR. The reactionmixture chosen for this was as follows: 20 ng of genomic DNA, 10 pmoleach of the two primers pamhnl4a (oBT2544) and pamhnl4e (oBT2543), 2 μlof dNTP mix (5 mM each), 1× Hotstar PCR buffer and 1.2 U of Hotstar DNApolymerase (Qiagen, Hilden, D). Amplification took place after a15-minute step at 95 DEG C with 30 cycles (1 min 94 DEG C, 30 sec 60 DEGC, 2 min 72 DEG C) and 15 min at 72 DEG C. The PCR product was purifiedtwice on Qiaquick (Qiagen, Hilden, D) columns and directly sequenced inorder to avoid sequence errors in cloned PCR products. The exons wereidentified in the resulting DNA sequence of the PCR fragment which is atotal of 2232 base pairs in length, and the protein sequence of thePaHNL4 isoenzyme was derived from the coding sequence. oBT2204 mandlp2f:5′-ACTACGAATTCGACCATGGAGAAATCAAC-3′ oBT2206 mandlp5r:5′-CACTGGAATTCAAAGAGCAACACTTATCCACGG-3′ oBT2543 pamhnl4e:5′-AAGAGGAACACTTAGCCACG-3′ oBT2544 pamhnl4a: 5′-CAACAATGTCCGCTGTAGTG-3′

EXAMPLE 2 Replacement of the Signal Sequence

2 variants were chosen as N terminus of the calculated mature protein(secreted protein after elimination of the signal peptide and of theadditional GluAlaGluAla sequence) in order to avoid accumulation ofincompletely processed enzyme in the interior of cells:

-   -   A) leucine as N-terminal amino acid as also occurs in the        wild-type sequence and which is regarded, according to the N-end        rule, as primary destabilizing amino acid (Varshavsky et al.,        Proceedings of the National Academy of Science of the United        States of America, 93(22), 12142-12149, 1996).    -   B) glutamine (mutation L1Q) as N-terminal amino acid which is        described as tertiary destabilizing amino acid (Varshavsky et        al., 1996).        PCR I:

The signal sequence of the alpha mating factor of Saccharomycescerevisiae was highly amplified from the template PPICZB (InvitrogenInc, San Diego, Ca, Cat. No. V19520). The PCR primers were constructedso that the EcoRI cleavage site of the Invitrogen plasmid at the 3′ endof the signal sequence was destroyed. This made it possible to clone theentire gene construct including the signal sequence via EcoRI cleavagesites into various Pichia expression vectors. The pairs of primers usedfor this purpose were alpha11/alpha21a and alpha11/alpha21aQ. Theprimers alpha11 and hnl5α21 comprise an EcoRI cleavage site. The primersalpha21a and alpha21aQ also comprised a DNA sequence region which codesfor the 5′ end of the mature PaHNL5 isoenzyme. The primer alpha21aQcomprised a sequence modification which leads to the mutation L1Q at theN terminus of the expected mature secreted protein. At the end of thealpha factor signal sequence there was a Kex2 cleavage site and aGluAlaGluAla sequence processed by Ste13.

The PCR was carried out in a 50 μl mixture (10 ng of template, 0.1 μM ofeach primer, 0.2 mM dNTPs, 5 μl of PCR buffer, 1 U of Pwo polymerasefrom Roche) in a thermocycler from Applied Biosystems (Forster City,CA). A denaturation step at 94° C. for 2 min was followed byamplification in 30 cycles (30 sec 94° C., 60 sec 62° C., 1 min 30 sec72° C.) and a concluding step at 72° C. for 7 min.

PCR II:

The hnl5 gene was highly amplified from the plasmid pHILDPaHNL5a(BT4256) using the pairs of primers hnl5α11/hnl5α21 andhnl5α11Q/hnl5α21. The primers hnl5α11 and hnl5α11Q also comprised a DNAsequence region which corresponded to the 3′ end of the fragment withthe alpha factor signal sequence (see above). The primer hnl5α21comprised an EcoRI cleavage site.

The PCR was carried out in a 50 μl mixture (10 ng of template, 0.1 μM ofeach primer, 0.2 mM dNTPs, 5 μl of PCR buffer, 1 U of Pwo polymerasefrom Roche) in a thermocycler from Applied Biosystems. A denaturationstep at 94° C. for 2 min was followed by amplification in 30 cycles (30sec 94° C., 60 sec 65° C., 3 min 30 sec 72° C.) and a concluding step at72° C. for 7 min.

Overlap Extension:

3 μl of each of the products from PCR I and PCR II were employed astemplate and simultaneously as primers for completion to give a coherentproduct. Extension took place in a 45 μl mixture with 5 μl of Pwo PCRbuffer, 0.2 mM dNTPs and 1 unit of Pwo polymerase (Roche, Mannheim, D).The mixture was heated at 94° C. for 2 min and then incubated in athermocycler with 10 cycles at 94° C. for 30 sec and at 72° C. for 3min.

PCR III:

The product from the overlap extension was amplified by using theprimers alpha11 and hnl5α21. 5 μl of primer mix (3 μl of water and 1 μlof each of the primers alpha11 and hnl5α21, the concentration of theprimers being 5 μM) were added to the overlap extension PCR mixture, andthe product was amplified with 20 cycles (30 sec 94° C., 45 sec 62° C.,4 min 72° C.). Finally, incubation took place at 72° C. for 7 min. ThePCR product was purified by the Qiaquick purification protocol of Qiagen(Hilden, D), cut with EcoRI and cloned into the vector pHILD2(Invitrogen, San Diegeo, CA).

Primer Sequences: oBT2835 alpha11:5′-tcttcgaagaattcacgATGAGATTTCCTTCAATTTTTACTGC-3′ oBT2841 alpha21a:5′-gaagtattggcaagAGCTTCAGCCTCTCTTTTCTCG-3′ oBT2843 alpha21aQ:5′-gaagtattggcttgAGCTTCAGCCTCTCTTTTCTCG-3′ oBT2837 hn15α11:5′-agagaggctgaagctCTTGCCAATACTTCTGCTCATG-3′ oBT2842 hn15α11Q:5′-agagaggctgaagctCAAGCCAATACTTCTGCTCATG-3′ oBT2838 hn15α21:5′-atggtaccgaattcTCACATGGACTCTTGAATATTATGAATAG-3′

Extensions are in lower case.

The resulting plasmids were called pHILDPaHNL5α (BT4338) andpHIL/PaHNL5α_L1Q (BT4339). Transformation into Pichia pastoris tookplace by the standard Invitrogen procedure.

Sterile toothpicks were used to inoculate deep well culture plates within each case approximately 2000 transformants, which were cultured forthe screening for active transformants.

EXAMPLE 3 Culturing of Pichia pastoris Transformants and production ofPaHNL5 Variants

a) Microcultures in Deep Well Plates

250 μl of BM0.5G medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/lyeast nitrogen base, 5 g/l glycerol, 0.8 mg/l biotin) in 2 ml deep wellplates were inoculated with single colonies of transformants and shakenat 28° C. and 340 rpm. Induction of expression via the AOX1 promotertook place by adding 250 μl of BMM2 medium (0.2 M potassium phosphate,pH 6.0; 13.4 g/l yeast nitrogen base, 20 ml/l methanol, 0.8 mg/l biotin)after 60-70 hours. Further methanol additions took place after 10, 24and 48 hours by adding in each case 50 μl of BMM10 (0.2 M potassiumphosphate, pH 6.0; 13.4 g/l yeast nitrogen base, 100 ml/l methanol, 0.8mg/l biotin).

About 72 hours after the induction, the cells were spun down and theculture supernatant was employed directly, diluted, or concentrated byultrafiltration through Vivaspin 30 kDa exclusion membranes fromSartorius (Göttingen, D) for measuring the enzymic activity.

b) “Scale Up” in Shaken Flasks

225 ml of BM0.5G medium (0.2 M potassium phosphate, pH 6.0; 13.4 g/lyeast nitrogen base, 5 g/l glycerol, 0.8 mg/l biotin) in 2 liter flaskswith baffles were inoculated with a large single colony and shaken at28° C. and 120 rpm. Induction of expression via the AOX1 promoter tookplace by adding 25 ml of BMM10 medium (0.2 M potassium phosphate, pH6.0; 13.4 g/l yeast nitrogen base, 100 ml/l methanol, 0.8 mg/l biotin)after 60-70 hours. Further additions of 2.5 ml of methanol per shakenflask (250 ml) took place after 10, 24 and 48 hours.

About 72 hours after the induction, the cells were spun down and theculture supernatant was employed directly, diluted or concentrated byultrafiltration through 30 kDa exclusion membranes for measuring theenzymic activity.

EXAMPLE 4 Site-Specific Mutagenesis

10 ng of the expression plasmid pHILDPaHNL5α_L1Q (PaHNL5 with alphafactor signal sequence) were employed as template for the mutagenesisreaction using the Quik Change XL Site Directed Mutagenesis Kit fromStratagene (Cat. #200516). 200 ng of each of the mutagenesis primerswere employed for the reaction. The following temperature program wasused:

-   A) denaturation at 95° C. for one minute-   B) 18 cycles with 50 sec at 95° C., 50 sec at 60° C. and-   20 min at 68° C.-   C) extension for 7 min at 68° C.

The template DNA was digested off with DpnI, as described in the kitprotocol, and 2 μl of the mixture were employed as described fortransforming ultracompetent E. coli XL 10 gold cells. Plasmid DNA wasprepared from the transformants and sequenced. Plasmids from mutantshaving the correct sequence in the region of the coding DNA insert werereplicated and transformed into Pichia pastoris GS115 with the aid ofthe standard Invitrogen procedure.

Several hundred histidine-autotrophic Pichia transformants werecultivated as described above in deep well plates, and the activity ofthe culture supernatants was determined with racemic mandelonitrile in96-well plates. Clones having in each case the highest enzymic activityof the individual mutants were selected for shaken flask experiments.The enzymic activity of the culture supernatants was determined usingthe substrate mandelonitrile (DSM Fine Chemicals Linz, A).

The following mutations were carried out:

A111, A111L, A111V, F72A, L331A and L343A, and as 5 comparativeexperiment V317A and V317G

PCR primers for the site-specific mutagenesis: oBT2966 oPaHNL5A111Gf:5′-GTGGCACGACCATAATCAATGGAGGCGTCTACGCCAGAGCTAAC-3′ oBT2967oPaHNL5A111Gr: 5′-GTTAGCTCTGGCGTAGACGCCTCCATTGATTATGGTCGTGCCAC-3′oBT3080 oPaHNL5A111Lf 5′ GCACGACCATAATCAATGCTTGCGTCTACGCCAGAGCTAAC 3′oBT3081 oPaHNL5A111Lr 5′ GTTAGCTCTGGCGTAGACGCAAGCATTGATTATGGTCGTGCCAC 3′oBT3078 oPaHNL5A111Vf 5′ GTGGCACGACCATAATCAATGGTTGCGTCTACGCCAGAGCTAAC 3′oBT3079 oPaHNL5A111Vr 5′ GTTAGCTCTGGCGTAGACGCAACCATTGATTATGGTCGTGCCAC 3′oBT2983 oPaHNL5V317(A,G)f: 5′TCCAATTGAAGCCTCTGTTGSAACTGTTTTAGGCATTAGAAGTG 3′ oBT2984oPaHNL5V317(A,G)r: 5′ CTAATGCCTAAAACAGTTSCAACAGAGGCTTCAATTGGATTTGG 3′oBT3017 oPaHNL5F72Af: 5′ CACGTTGACTGCAGATGGGGCTGCATATAATCTGCAGCAACAAG 3′oBT3018 oPaHNL5F72Ar: 5′ CTTGTTGCTGCAGATTATATGCAGCCCCATCTGCAGTCAACGTG 3′oBT3019 oPaHNL5L343Af: 5′ CCACTCCACCCTTTAGTGCTTTTCCTACAACATCTTACCCCCTC3′ oBT3020 oPaHNL5L343Ar: 5′AAGATGTTGTAGGAAAAGCACTAAAGGGTGGAGTGGAAAATGGC 3′ oBT3021 oPaHNL5L331Af:5′ AGTGATTATTATCAAGTTTCTGCCTCAAGCTTGCCATTTTCCAC 3′ oBT3022 oPaHNL5L331Ar5′ GGAAAATGGCAAGCTTGAGGCAGAAACTTGATAATAATCACTTC 3′

Transformants which expressed the PaHNL5 mutants were isolated, and theregion of the mutation checked by sequencing by the colony PCR method.

Table 1 shows an overview of the respective expression strains of themutants: TABLE 1 modifications introduced into the pahnl5 gene PichiaPlasmid for Signal clone Pichia trans- AA DNA sequence Pichia clone nameNo. formation exchange sequence plant PaHNL5 GS115pHILDPaHNL1a37 BT2578BT4256 signal Alpha factor GS115pHILDPaHNL5alpha_G13 BT2617 BT4338 Alphafactor GS115pHILDPaHNL5alphaLIQ_H21 BT2620 BT4339 LIQ CTT→CAA Alphafactor GS115pHILDPaHNL5alpha_LIQ, BT2621 BT4345 LIQ CTT→CAA A111G (=sp1a8F9) A111G GCA→GGA Alpha factor GS115pHILDPaHNL5alpha_LIQ, BT2623BT4375 LIQ CTT→CAA V317A (= spA2.2) V317A GTA→GCA Alpha factorGS115pHILDPaHNL5alpha_LIQ, BT2624 BT4376 LIQ CTT→CAA V317G (= spAa)V317G GTA→GGA Alpha factor GS115pHILDPaHNL5alpha_LIQ, BT2632 BT4400 LIQCTT→CAA F72A F72A TTT→GCT Alpha factor GS115pHILDPaHNL5alpha_LIQ, BT2633BT4401 LIQ CTT→CAA L331A L331A CTG→GCC Alpha factorGS115pHILDPaHNL5alpha_LIQ, BT2634 BT4402 LIQ CTT→CAA L343A L343A CTT→GCTAlpha factor GS115pHILDPaHNL5alpha_LIQ, BT2635 BT4403 LIQ CTT→CAA F72A,L331A F72A TTT→GCT L331A CTG→GCC Alpha factor GS115pHILDPaHNL5alpha_LIQ,BT2636 BT4404 LIQ CTT→CAA F72A, L331A, L343A F72A TTT→GCT L331A CTG→GCCL343A CTT→GCT Alpha factor GS115pHILDPaHNL5alpha_LIQ, BT2637 BT4405 LIQCTT→CAA A111L A111L GCA→CTA Alpha factor GS115pHILDPaHNL5alpha_LIQ,BT2638 BT4406 LIQ CTT→CAA A111V A111V GCA→GTT

EXAMPLE 6 Purification and Characterization of Prunus amygdalus EnzymeVariants

The specific activity of the respective mutants with differentsubstrates was determined by carrying out several shaken flask cultureswith each of the expression clones. The culture supernatant wasconcentrated by ultrafiltration (30 kDa cutoff) using 20 ml Vivaspin PEScentrifugation columns from Sartorius (Gottingen, D) and then purifiedby chromatography.

Before the purification, the concentrated culture supernatant wasequilibrated with the low-salt binding buffer A by repeated dilution andconcentration with binding buffer A (20 mM citrate-phosphate buffer, pH5.5) in 30 kDa ultrafiltration centrifugation modules (Vivaspin,Sartorius), and then purified on a Q-Sepharose Fast Flow (QFF) anionexchange column with a column volume of 10 ml in an AKTApurifier 10 FPLCsystem from Amersham Biosciences UK Limited (Buckinghamshire, GB).Elution took place with elution buffer B (20 mM citrate-phosphatebuffer+1M NaCl, pH 5.5), using the following gradient profile for thedifferent variants of PaHNL5 from heterologous production with Pichiapastoris:

One column volume as washing step proved to be ideal for washing out allunbound protein constituents. The concentration of buffer B (elutionbuffer: 20 mM citrate-phosphate buffer, 1M NaCl, pH 5.5) was raised inhalf a column volume to 4% and subsequently increased to 48% in afurther column volume. The next step was to increase the concentrationof elution buffer B to 70%, using 1½ column volume in this case.

Finally, the concentration was raised to the maximum of 100% in onecolumn volume and was in conclusion left thereat for a further columnvolume (washing step without fractionation).

Those fractions which ought, according to evaluation of thechromatogram, to contain protein (depending on the peak position)underwent determination of the protein content using the Biorad(Hercules, Ca) protein assay (Bradford method) and of the enzymicactivity using the substrate mandelonitrile. The 2-3 fractions with thehighest activity were pooled and employed for analyzing the enzymecharacteristics. The protein concentration was carried out with a Biorad(Hercules, Ca) protein assay (Bradford). The standard used for producinga calibration line was native PaHNL from Sigma (M-6782 Lot 41H4016).

The culture supernatants were concentrated ˜20-fold by cross-flowfiltration and then purified by chromatography. Samples were taken ofthe purified enzymes and loaded directly onto a gel (protein gel NuPAGE4-12% bis gel 1 mm×17 well; Invitrogen), or ˜500 ng were deglycosylatedwith endoglycosidase H (#P0702L, NEB) (according to the proceduresupplied) and then loaded. The standard used was “SeeBlue Plus2Pre-Stained Standard” from Invitrogen (Carlsbad, USA).

To compare the substrate specificities, the protein concentration of thepurified enzymes and the protein content in the culture supernatant wasmeasured using the Biorad protein assay (Hercules, Ca), and the specificactivities were compared with racemic mandelonitrile photometrically andwith 2-, 3- and 4-chlorobenzaldehyde by GC:

For this purpose, 15 mmol of substrate were dissolved in 2.1 ml oftert-butyl methyl ether (MTBE). 0.25 mg of the appropriate PaHNL wasdiluted with 50 mM K2HPO4/citrate buffer of pH 3.4 to a final volume of3.7 ml, the buffer was again adjusted to pH 3.4 and then mixed with thesubstrate in MTBE in 20 ml glass vials. The solution was cooled to 10°C., and 1.2 ml of HCN was added with a syringe and stirred at 10° C. ona magnetic stirrer to form an emulsion. Samples were taken at varioustimes, derivatized with acetic anhydride in the presence of pyridine anddichloromethane, and analyzed by GC on a cyclodextrin column(CP-Chirasil-Dex CB) or by HPLC. TABLE 2 specific activities(μmol/min/mg) 2-Cl- 4-Cl- Mandelonitrile benzal- 3-Cl- benzal- Mutant(cleavage) dehyde benzaldehyde dehyde WT 295 +/− 30 n.d. n.d. n.d.AlphaWT 325 +/− 30 92 +/− 30  260 +/− 120 514 +/− 80 A111G  8 +/− 3 402+/− 90  482 +/− 60 458 +/− 50 V317G   6 +/− 1.5 <10 226 +/− 40  21 +/−10 (racemic)n.d not determinedWT: PaHNL5 expressed with native vegetable signal sequenceAlphaWT: PaHNL5 with S. cerevisiae alpha mating factor preproleader,EAEA sequence and L1Q mutationA111G: as AlphaWT with additional A111G mutationV317G: as AlphaWT with additional V317G mutation (comparativeexperiment)

The measurements revealed that the specific activity of the A111G mutantwith the substrate (R)-2-chloro-mandelonitrile was about 3-5 timeshigher than that for the recombinant wildtype WT and AlphaWT isoenzymesof PaHNL5. The activity with 3-chlorobenzaldehyde was also higher withthe A111G mutant than with AlphaWT.

EXAMPLE 7 Preparation of Mutants A111G

A sufficient amount of enzyme for pilot conversions was prepared fromthe improved clone Pichia pastoris GS115 pHILDPaHNL5alpha_L1Q,A111G(=BT2621) in a pilot fermentation.

8 flasks (2 l wide-neck) with baffles, each containing 250 ml of BMGmedium (according to the standard Invitrogen protocol), were inoculatedwith single colonies of the strain Pichia pastorisGS115pHILDPaHNL5alpha_L₁Q,A₁₁₁G and shaken (120 rpm) at 28° C. for 36hours. Chemicals 1-9, quantities for 20 liters, were brought to a totalweight of 15 kg with deionized water and introduced into a 40 lbioreactor (MBR, Oftringen, CH). Sterilization in situ was followed byadjustment of the pH of the medium to pH 5.0 with 28% ammonia through asterile feed pump. 200 ml of sterile-filtered “trace element solution”(together with vitamin H-biotin) were then introduced through a feedbottle into the bioreactor. A further 200 ml of the “trace elementsolution” were also added every second day until the end of thefermentation. Inoculation took place with 1.4 kg of preculture from theshaken flasks. The initial weight of the fermenter contents was about 15kg. With an operating temperature of 28° C., an aeration rate of 10-30liters of air/min and a stirring speed between 350 and 700 rpm, thepartial pressure of oxygen (pO2) was kept at a value >10% of thesaturation concentration. After 27 hours, the biomass had grown to avalue of 122.8 g/l wet weight of cells or 30 g/l cell dry weight (CDW).From this time onwards, about 70 g of sterile glycerol was added insmall portions per hour. In this linear phase of growth it was possibleto reach a biomass concentration in the region of 100 g/l CDW in aperiod of 60 hours.

Thereafter the third phase was initiated by inducing expression byadding methanol. The methanol content in the culture broth was in thiscase adjusted to a value of 0.8-1% by weight. As the oxygen consumptionincreased during the fermentation, methanol (0.8-1 percent by weight)was added anew in each case. The increase in the enzymic activity wasfollowed by photometric determination of the activity in the culturesupernatant of samples which were taken approximately every 12 hoursfrom the fermenter. After methanol induction for 210 hours, the increasein enzymic activity was very small and the cells were harvested bycentrifugation at 4000 g for 20 min twice, and the culture supernatantwas collected. The enzymic activity in the culture supernatant aftercentrifugation was 3.3 U/ml (standard HNL assay with rac.mandelonitrile), resulting in an enzyme yield of about 22 000 U for anoverall yield of about 6.5 liters of culture supernatant from 14.3 kg offermenter contents.

The supernatant was purified from remaining cell material by 0.2 ucrossflow filtration (VIVASCIENCE Vivaflow 50 from Sartorius, Gottingen,D). Concentration took place by crossflow ultrafiltration with Sartorius30 kDa 50 cm2 cutoff modules. Enzyme preparations with 24.5 U/ml and 57U/ml were prepared in this way for pilot experiments on cyanohydrinsynthesis. Since Pichia pastoris secretes scarcely any of its ownproteins into the culture supernatant, the enzyme produced andconcentrated in this way was also very pure by comparison with plantenzyme preparations.

The following chemicals were used to prepare the culture medium (amountper liter): 1. 85% ortho-phosphoric acid   35 ml 2. CaSO₄ 0.68 g 3.K₂SO₄ 18.8 g 4. MgSO₄.7H₂O 13.4 g 5. KOH  5.7 g

(Chemicals 1 to 5 in analytical quality)

-   6. Glycerol, technical quality. 50 ml-   7. Deionized water, conductivity 5.5-9.1 μS/cm-   8. Antifoam 10% Acepol 83E (Carl Becker Chemie GmbH, Hamburg, D) 1    ml-   9. 25% ammonia, technical quality 70 g/l

Trace elements and vitamin H (all chemicals in analytical quality): 10.Biotin  0.8 mg 11. CuSO₄.5H₂O 24.0 mg 12. KI 0.32 mg 13. MnSO₄.H₂O 12.0mg 14. Na₂MoO₄.2 H₂O  0.2 mg 15. H₃BO₃ 0.08 mg 16. CoCl₂  2.0 mg 17.ZnSO₄.7H₂O   80 mg 18. Fe(II)SO₄.7H₂O  260 mg

EXAMPLE 8 Preparative Conversions with Benzaldehyde and2-chlorobenzaldehyde

The enzyme properties in preparative synthesis were analyzed byconverting 150 mmol of substrate in a reactor.

150 mM substrate were diluted or dissolved with 21 ml of MTBE. 5 mg of“PaHNL5alpha_L1Q,A111G” enzyme (A111G mutant) were diluted with 50 mMK2HPO4/citrate, pH 3.4, to a volume of 37.5 ml and adjusted to pH 3.4with 10% strength citric acid. This aqueous phase was added to theorganic phase and stirred in a 100 ml Schmizo KPG stirrer for 5 min. Thetemperature was kept at 10° C., and HCN was metered in by means of aperfuser pump for 1 hour. The reaction was stirred at 900 rpm at 10° C.For workup, the reaction solution was diluted with 140 ml of MTBE,stirred for 5 min and, after 10 min, the phases were separated. Theaqueous phase was extracted once more with 40 ml of MTBE. Afterspontaneous phase separation, the organic phases were combined,stabilized with citric acid and evaporated. Analysis by GC was carriedout as described above.

The conversions gave after 7 hours a yield of 95.1% 2-chlorobenzaldehydecyanohydrin with an ee of 95.7% and a yield of more than 99%mandelonitrile with an ee of >99%.

EXAMPLE 9 Enzyme Stability at Low pH

The enzyme samples of commercially available native PaHNL from almondkernels (Sigma) and the A111G mutants were diluted in 50 mMcitrate-phosphate buffer of pH 6.5 until, after a further 1:70 dilution,an increase of about 100 mOD was to be expected in the photometricdetermination of activity at 280 nm by the standard HNL assay withracemic mandelonitrile. 150 μl of these dilutions were transferred into900 μl of 0.1M phosphate buffer with appropriately adjusted pH (dilution1:7) and then, at various times after incubation at 22° C., 100 μl ofthese dilutions were employed for the determination of activity (100 μlof enzyme solution, 700 μl of 1M phosphate-citrate buffer of pH 5.0 and200 μl of 60 mM mandelonitrile in 3 mM citrate-phosphate buffer of pH3.5). The pH stability of the mutant PaHNL5 alpha_L1Q, A111G (A111G) atpH 2.6 compared with commercially available native PaHNL from almondkernels (Sigma) is evident from FIG. 1.

1. An R-hydroxynitrile lyase from the Rosaceae family having improvedsubstrate acceptance and increased stability, characterized in that inthe active center of the R-hydroxynitrile lyase there is replacementeither of a) an alanine residue by glycine, valine, leucine, isoleucineor phenylalanine or b) a phenylalanine residue by alanine, glycine,valine, leucine or isoleucine or c) a leucine residue by alanine,glycine, valine, isoleucine or phenylalanine or d) an isoleucine residueby alanine, glycine, valine, leucine or phenylalanine.
 2. AnR-hydroxynitrile lyase as claimed in claim 1, characterized in thatthere is replacement in the active center of the R-hydroxynitrile lyaseeither of an alanine residue by glycine, valine or leucine, of aphenylalanine residue by alanine, leucine or glycine, or of a leucineresidue by alanine, glycine or phenylalanine.
 3. An R-hydroxynitrilelyase as claimed in claim 1, characterized in that the replacements iscarried out in the active center of R-hydroxynitrile lyases from Prunusamygdalus, Prunus serotina, Prunus laurocerasus, Prunus lyonii, Prunusarmaniaca, Prunus persica, Prunus domestica or from Malus communis, andof recombinant R-hydroxynitrile lyases thereof or of tunnel mutants ofthe R-hydroxynitrile lyases listed above, in which one or more bulkyamino acid residues within the hydrophobic channel leading to the activecenter are replaced by less bulky amino acid residues.
 4. AnR-hydroxynitrile lyase as claimed in claim 1, characterized in that theR-hydroxynitrile lyase to be modified is in the form of the completesequence or of a sequence which has been modified by exchange of thefirst amino acid(s) or of a sequence truncated by deletion of the firstamino acid(s) or of a sequence extended by attachment of further aminoacids.
 5. An R-hydroxynitrile lyase as claimed in claim 1, characterizedin that before the mutation in the active center the natural orvegetable signal sequence is replaced by the signal sequence of thealpha mating factor from Saccharomyces cerevisiae, Saccharomycescerevisiae invertas, Pichia killer toxin signal sequence, a-amylase,Pichia pastoris acid phosphatase, Phaseolus vulgaris aggiutini;glycoamylase signal sequence from Aspergillus niger, glucose oxidasesignal sequence from Aspergillus niger, Sec10 signal sequence fromPichia pastoris, signal sequence of the 28 kD subunit of the killertoxin from Klyveromyces lactis or the BSA signal sequence, or by arecombinant signal sequence thereof or by one of the abovementionedsignal sequences with point mutation.
 6. An R-hydroxynitrile lyase asclaimed in claim 1, characterized in that preparation takes place bysite-specific mutagenesis with subsequent secretory expression in amicroorganism from the group of Pichia pastoris, Saccharomycescerevisiae or Escherichia coli, Bacillus subtilis, Klyveromyces lactis,Aspergillus niger, Penicillium chrysogenum, Pichia methanolica, Pichiapolyrnorpha, Phichia anomala, or Schizosaccharomyces pombe.
 7. AnR-hydroxynitrile lyase as claimed in claim 1, characterized in that thereplacement is carried out in the active center of a nativeR-hydroxynitrile lyase from Prunus amygdalus, Prunus domestica or fromPrunus serotina or of a recombinant R-hydroxynitrile lyase from thegroup of PdHNL1, PaHNL1, PaHNL2, PaHNL3, PaHNL4 or PaHNL5, each of whichmay optionally have a mutation in the hydrophobic channel leading to theactive center.
 8. An R-hydroxynitrile lyase as claimed in claim 1,characterized in that there is replacement in the active center of therecombinant R-hydroxynitrile lyases PaHNL5, which may optionally alsohave a mutation in the hydrophobic channel leading to the active center,either of a) the alanine residue at position 111 by glycine, valine orleucine or b) the phenylalanine residue at position 72 by alanine orleucine or c) the leucine residue at position 331 or 343 by alanine orglycine.
 9. A method for preparing enantiopure R- or S-cyanohydrinscomprising using the R-hydroxynitrile lyases as claimed in claim
 1. 10.A process for preparing enantiopure R- or S-cyanohydrins, characterizedin that aliphatic, aromatic or heteroaromatic aldehydes or ketones areconverted n the presence of a cyanide group donor with anR-hydroxynitrile lyases as claimed in claim 1 in an organic, aqueous or2-phase system or in emulsion or undiluted at a temperature of from −10°C. to +50° C. and at a pH of from 1.8 to
 7. 11. A process for preparingenantiopure R- or S-cyanohydrins, characterized in that aliphatic,aromatic or heteroaromatic aldehydes or ketones having bulkysubstituents are converted with an R-hydroxynitrile lyase as claimed inclaim 1, in which one of the amino acids alanine, valine, leucine,isoleucine or phenylalanine in the active center has been replaced by arespectively smaller amino acid residue, and in that aliphatic, aromaticor heteroaromatic aldehydes or ketones having small substituents areconverted with the an R-hydroxynitrile lyases in which one of the aminoacids alanine, valine, leucine, isoleucine or phenylalanine in theactive center has been replaced by a respectively, larger amino acidresidue.
 12. An R-hydroxynitrile lyase as claimed in claim 8,characterized in that the R-hydroxynitrile lyase to be modified is inthe form of the complete sequence or of a sequence which has beenmodified by exchange of the first amino acid(s) or of a sequencetruncated by deletion of the first amino acid(s) or of a sequenceextended by attachment of further amino acids.
 13. An R-hydroxynitrilelyase as claimed in claim 8, characterized in that before the mutationin the active center the natural or vegetable signal sequence isreplaced by the signal sequence of the alpha mating factor fromSaccharomyces cerevisiae, Saccharomyces cerevisiae invertas, Pichiakiller toxin signal sequence, α-amylase, Pichia pastoris acidphosphatase, Phaseolus vulgaris aggiutini; glycoamylase signal sequencefrom Aspergillus niger, glucose oxidase signal sequence from Aspergillusniger, Sec10 signal sequence from Pichia pastoris, signal sequence ofthe 28 kD subunit of the killer toxin from Klyveromyces lactis or theBSA signal sequence, or by a recombinant signal sequence thereof or byone of the abovementioned signal sequences with point mutation.